getyourscienceonfandomcom-20200216-history
Smash N Grab Yeast Genomic DNA Prep
Smash N Grab yeast genomic DNA Prep 1. Grow small cultures (usually 3ml) overnight at 30C in YPD. 2. Fill a 1.5ml microfuge tube with the culture and collect the cells by 1 min centrifugation in a microfuge. 3. Aspirate the supernatant off. 4. Add 200ul of SnG Lysis Buffer Triton X-100, 1% SDS, 100mM NaCl, 10mM Tris-Cl(pH 8), 1mM Na2-EDTA and then briefly vortex the tube to suspend the pellet- make or order more SnG if there is none on your bench, or talk to Dan. Add 200ul of phenol:chloroform:isoamyl alcohol (25:24:1), which is on the bottom shelf of the upright -20C in J329. Add 0.3g of acid-washed glass beads (1 "scoop"), there are some on Dan's shelf or ask for your own supply. NOTE BENE: Phenol is a uniquely hazardous substance. If you have never worked with phenol, have some one in the lab go over proper precautions, and proper disposal, one-on-one in person before using it for anything at all. 5. Vortex hard for 5 minutes. 6. Centrifuge for 10 minutes at top speed in a microfuge. 7. Carefully remove ~160ul clearest supernatant and add to 400ul 100% EtOH in a fresh microfuge tube. 8. Allow a 5 min incubation at room temp. Centrifuge again for 5 min, top speed. Remove supernatant. 9. Wash pellet in 500 ul 70% EtOH. Dry on bench. Options: A) If you need the samples to PCR TODAY, open the microfuge tubes and set them upright, upside down on a paper towel. The absorptive action of the paper towel will help draw out the moisture faster than air drying, your sample will be dry in around 20 minutes. If you think it is not dry enough, then it isn't. It costs less to wait another ten minutes than it does to have to redo the SnG. B) If the samples are not needed today, then dry them overnight. Leave the tubes standing upright in the tube rack, open the caps, and put a piece of parafilm over the whole rack, then move it out of the way on your bench. 10. Resuspend in 50ul H2O. Flick the tube, do not vortex it! A NOTE ON ADJUSTING PH OF SOLUTIONS: For the love of all that is holy please do not use the pH meter without getting a tutorial first; there are people who loooooooooove to have very long meetings / lectures about imagined misuse of the pH meter, and to be fair this is because it is the pH meter we currently have is a very temperamental and unfortunate design, which is very easy to break and has been broken previously more than once. If I have to hear some bullshit about the pH meter because of you I will be officially pissed. For 50ml of lysis buffer: 2.5ml 20% SDS P/N: Sigma L-5750 10ml 10% Triton X-100 P/N: Sigma-Aldrich T8532-500ML ''' 1ml 5M NaCl '''P/N: EMD SX0420-3 0.5ml 1M Tris-Cl, pH 8 'P/N: Sigma-Aldrich T1503-500G ' 0.25 ml 0.2 M EDTA 'P/N: OmniPur 4050 '